Differential Expression of VEGF Isoforms and VEGF ...

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chondroblastoma, a chondromyxoid fibroma, a grade 2 chondrosarcoma, a mesenchymal chondrosarcoma and four osteosarcomas. Total RNA was extracted ...
ANTICANCER RESEARCH 25: 955-958 (2005)

Differential Expression of VEGF Isoforms and VEGF Receptors in Cartilaginous Tumors KIMINORI YUKATA, YOSHITO MATSUI, TOMOHIRO GOTO, TAKAHIRO KUBO and NATSUO YASUI

Department of Orthopaedics, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan

Abstract. Vascular endothelial growth factor (VEGF), which is known to have at least five isoforms and four receptors, is integral to tumor-induced neovascularization. In order to determine whether VEGF signaling is modulated in the development of cartilaginous tumors, we analyzed osteochondrogenic tumors for the expression of VEGF isoforms and VEGF receptors using the reverse transcriptionpolymerase chain reaction method. Although mRNA for VEGF121, VEGF165, VEGFR-1 and NRP-2 was widely expressed, mRNA expression for VEGF189, VEGFR-2 and NRP-1 was linked to a malignant phenotype such as local invasion. These results indicated that VEGF signaling was fine-tuned by the differential expression of the VEGF isoforms and VEGF receptors. Cartilage is an avascular tissue composed of highly differentiated resident cells (i.e. chondrocytes) and extracellular matrix. During development and in some pathological conditions such as inflammation and oncogenesis, however, cartilage is associated with neovascularization, where vascular endothelial growth factor (VEGF) signaling is suggested to be integral for the cells to exert biological actions (1). For example, osteoarthritic chondrocytes express VEGF, which induces matrix metalloproteinase expression and degrades cartilage extracellular matrix (2). VEGF shows a strong angiogenic activity through its chemotactic and/or mitotic action on cells. The primary transcript of VEGF undergoes alternative mRNA splicing, which generates five isoforms, VEGF121, VEGF145,

Correspondence to: Yoshito Matsui, Department of Orthopaedics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Tel: +1-88-633-7240, Fax: +1-88-633-0178, e-mail: [email protected] Key Words: Alternative splicing, angiogenesis, cartilaginous tumors, VEGF.

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VEGF165, VEGF189 and VEGF206, containing 121, 145, 165, 189 and 206 amino acid residues, respectively (2). In addition, there are four known VEGF receptors, VEGFR-1, VEGFR-2, NRP-1 (neuropilin-1) and NRP-2, which are genetically distinct (2,3). Therefore, it is plausible to speculate that VEGF signaling is fine-tuned by having these isoforms and receptors. Among cartilaginous tumors, malignant tumors are associated with more neovascularization while benign tumors are associated with less (4, 5). A previous immunohistochemical report showed that malignant cartilaginous tumors expressed VEGF while benign tumors did not. However, the fine-tuning of the VEGF signaling in this tumor-induced neovascularization is not clear. In the present study, using the reverse transcription (RT) polymerase chain reaction (PCR) method, we examined the expression of VEGF isoforms as well as VEGF receptors in a panel of osteochondrogenic tumors.

Materials and Methods Tissues from ten osteochondrogenic tumors were obtained at biopsy or at surgery under informed consent. The tumor types were histologically defined as an osteochondroma, an enchondroma, a chondroblastoma, a chondromyxoid fibroma, a grade 2 chondrosarcoma, a mesenchymal chondrosarcoma and four osteosarcomas. Total RNA was extracted from each tumor specimen and normal human cartilage by a standard method (6). After DNase (Invitrogen, Carlsbad, CA, USA) treatment, singlestranded cDNA was synthesized using 3 Ìg of each RNA sample, 100 ng of random primers and 200 U of SUPERSCRIPT II RNase H- Reverse Transcriptase in a total volume of 20 Ìl (Invitrogen). cDNA fragments corresponding to the alternatively spliced VEGF transcripts were amplified by PCR using a primer set spanning all the alternative exons (2). cDNA fragments for VEGFR-1, VEGFR2, NRP-1 and NRP-2 were also amplified using primer sets described previously (2, 3). Amplification was performed with 18 Ìl of PCR Supermix (Invitrogen) in a total volume of 21 Ìl containing 10 pmol of each primer and 1 Ìl of cDNA. The PCR conditions were as follows: initial denaturation for 1 minute at 95ÆC, 32 cycles of 30 seconds each at 95ÆC, 60ÆC and 72ÆC, and final extension for 7 minutes at 72ÆC. A 452-bp fragment of glyceraldehyde-3-

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ANTICANCER RESEARCH 25: 955-958 (2005)

Figure 1. A: Schematic representation of VEGF isoforms. VEGF189 contains exon 1 through exon 5, exon 6a, exon 7 and exon 8. VEGF165 lacks exon 6a and VEGF121 lacks exon 6a as well as exon 7. B: RT-PCR analysis of VEGF isoforms and VEGF receptors in osteochondrogenic tumors. Abbreviations: CA; cartilage, OC; osteochondroma, EC; enchondroma, CB; chondroblastoma, CMF; chondromyxoid fibroma, CS; grade 2 chondrosarcoma, MCS; mesenchymal chondrosarcoma, OS; osteosarcoma.

phosphate dehydrogenase (GAPDH) cDNA was also amplified as a control. Aliquots of the PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. Each band was identified by direct DNA sequencing using the PRISM BigDye Terminator Cycle sequencing Kit (Applied Biosystems, Foster City, CA, USA). The reproducibility of the PCR experiment was confirmed in triplicate experiments.

Results and Discussion RT-PCR analysis demonstrated that the predominant VEGF mRNA isoform in human normal cartilage was VEGF121 (Figure 1). VEGFR-1 and NRP-2 mRNAs were also expressed in normal cartilage. Osteochondroma and enchondroma, which are highly-differentiated benign cartilaginous tumors (7), expressed VEGF121 and VEGF165 isoforms. In osteochondroma, mRNA for VEGFR-1 was also detectable. Chondroblastoma expressed VEGF121 mRNA together with VEGFR-1 and NRP-2 mRNA, although this benign cartilaginous tumor lacks differentiated cartilaginous phenotype (8, 9). Chondromyxoid fibroma is another benign cartilaginous tumor with aggressiveness for bone destruction, which showed VEGF189 mRNA isoform in addition to VEGF121 and VEGF165 isoforms. Furthermore, mRNAs for VEGFR-2 and NRP-1 were detectable together with VEGFR-1 and NRP-2. Grade 2 chondrosarcoma and mesenchymal chondrosarcoma, malignant cartilaginous tumors, also expressed mRNAs for VEGF121, VEGF165 and VEGF189 isoforms, as well as VEGFR-1, VEGFR-2, NRP-1 and NRP-2. Osteosarcoma, a malignant bone tumor, showed the same expression pattern of VEGF isoforms and VEGF receptors as chondromyxoid fibroma and chondrosarcoma.

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It is known that the expression of VEGF and its receptor genes are one of the characteristics of cartilaginous tumors (4, 5) and, to our knowledge, this is the first report of mRNA splicing of VEGF transcripts in such tumors. The results showed that the VEGF isoforms and the VEGF receptors were differentially expressed in a panel of osteochondrogenic tumors. Although mRNA for VEGF121, VEGF165, VEGFR-1 and NRP-2 was widely expressed, mRNA expression for VEGF189, VEGFR-2 and NRP-1 was linked to a malignant phenotype, i.e., hypervascularity associated with local invasion in chondrosarcomas and osteosarcomas. Chondromyxoid fibroma also expressed VEGF189, VEGFR-2 and NRP-1 mRNA, although this tumor shows a benign clinical course. This may be related to the locally aggressive bone destruction, which is a characteristic of chondromyxoid fibroma, probably by inducing a variety of matrix metalloproteinases (2). In addition to VEGF121 mRNA, VEGF165 mRNA was significantly expressed in osteochondroma and enchondroma. However, these tumors contained less mRNA for VEGF receptors, suggesting locally conservative phenotype. Chondroblastoma showed a VEGF and VEGF receptor mRNA expression pattern, which was similar to that in normal cartilage. Again, this may reflect a lack of locally aggressive bone destruction phenotype. In conclusion, our findings indicated that cartilaginous tumors were under fine-tuning of VEGF signaling by differential expression of VEGF isoforms and VEGF receptors. Controlling the fine-tuning of VEGF signaling may prevent locally aggressive bone destruction in cartilaginous tumors.

Yukata et al: VEGF Isoforms and VEGF Receptors in Cartilaginous Tumors

Acknowledgements This work was supported in part by the Japan Society for the Promotion of Science (grant No. 16591496 and the 21st century COE Grant) and the Uehara Memorial Foundation, Japan.

References 1 Robinson CJ and Stringer SE: The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 114: 853-865, 2000. 2 Enomoto H, Inoki I, Komiya K, Shiomi T, Ikeda E, Obata K, Matsumoto H, Toyama Y and Okada Y: Vascular endothelial growth factor isoforms and their receptors are expressed in human osteoarthritic cartilage. Am J Pathol 162: 171-181, 2003. 3 Handa A, Tokunaga T, Tsuchida T, Lee YH, Kijima H, Yamazaki H, Ueyama Y, Fukuda H and Nakamura M: Neuropilin-2 expression affects the increased vascularization and is a prognostic factor in osteosarcoma. Int J Oncol 17: 291295, 2000. 4 Ayala G, Liu C, Nicosia R, Horowitz S and Lachman R: Microvasculature and VEGF expression in cartilaginous tumors. Hum Pathol 31: 341-346, 2000. 5 McGough RL, Aswad BI and Terek RM: Pathological neovascularization in cartilagionous tumors. Clin Orthop 397: 76-82, 2002.

6 Sambrook J, Fritsch EF and Maniatis T: Molecular Cloning: A Laboratory Manual. New York, Cold Spring Harbor Laboratory Press, 1989. 7 Aigner T, Dertinger S, Vornehm SI, Dudhia J, von der Mark K and Kirchner T: Phenotypic diversity of neoplastic chondrocytes and extracellular matrix gene expression in cartilaginous neoplasms. Am J Pathol 150: 2133-2141, 1997. 8 Matsui Y, Kimura T, Tsumaki N, Nakata K, Yasui N, Araki N, Hashimoto N, Uchida A and Ochi T: Splicing patterns of type XI collagen transcripts act as molecular markers for osteochondrogenic tumors. Cancer Lett 124: 143-148, 1998. 9 Matsui Y, Nakata K, Araki N, Ozono K, Fujita Y, Tsumaki N, Kawabata H, Yasui N, Sekiguchi K and Yoshikawa H: Alternative splicing of fibronectin transcript in osteochondrogenic tumors. Anticancer Res 21: 1103-1106, 2001.

Received November 3, 2004 Accepted February 7, 2005

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